Human pluripotent stem cells, such as human ES cells and human iPS cells, have attracted worldwide attention for their potential application to regenerative medicine. For culture and maintenance of human pluripotent stem cells in a pluripotent state, co-culture with feeder cells such as mouse or human fibroblasts is usually employed, but the use of feeder cells is a great restriction on clinical application of human pluripotent stem cells. In order to solve this problem, various adhesion proteins in place of feeder cells have been attempted for use as an extracellular matrix for human ES cells, and it is reported that Matrigel (trade name), which is composed of basement membrane components extracted and purified from mouse EHS sarcoma known to excessively produce basement membrane components, is highly effective in maintenance of pluripotency. However, since Matrigel is a mouse derived product and a complicated mixture of xenogeneic proteins and polysaccharides, it is not suitable for culture of human pluripotent stem cells in regenerative medicine.
For application of human pluripotent stem cells to regenerative medicine, cell culture must be performed in xeno-free conditions, in which the culture system does not contain any xenogeneic components, and in feeder-free conditions, in which feeder cells are not used. Based on these requirements, human vitronectin or human fibronectin has been attempted for use as a human extracellular matrix, but as compared with Matrigel, either of them are not superior in maintenance of undifferentiated state of human ES cells or in attachment efficiency thereof, and unsatisfactory in terms of quality, source material availability, safety, etc. Thus, in the development of extracellular matrices suitable for maintenance culture of human pluripotent stem cells, there are much to be improved, and development of a new human-pluripotent-stem-cell culture technology using a human extracellular matrix having a uniform chemical composition is strongly desired.
Laminin is a major cell-adhesion protein present in the basement membrane. Laminin is a large heterotrimeric glycoprotein consisting of three subunits termed α, β and γ chains, and has a molecular weight of 800,000 Da. The three subunits associate with each other through their coiled-coil domains in the C-terminal regions and assemble into a heterotrimer that is stabilized by disulfide bonds therein. The present inventors examined the type of integrins expressed on human ES cells, and reported that α6β1 integrin is a major adhesion receptor of human ES cells and that recombinant human laminins (particularly, laminin 332 composed of α3, β3 and γ2 chains, and laminin 511 composed of α5, β1 and γ1 chains) are effective for maintaining the pluripotency of human ES cells (see Non Patent Literature 1). The present inventors also reported that laminin 511 has a very high affinity for α6β1 integrin, and that the E8 fragment of laminin 511 has an α6β1 integrin-binding activity equivalent to that of the full-length laminin 511 (see Non Patent Literature 2).
However, since laminin binds to various cell surface molecules and extracellular matrix molecules, many technical problems must be overcome for preparation of a pure and homogeneous laminin free from impurities. In addition, since each of the three laminin subunits has a molecular weight of as large as 200,000 to 400,000 Da, the heterotrimeric molecule assembled from these subunits is not easy to be expressed as a recombinant protein, and its yield is low.